Pseudomonas syringae pv. coryli, the causal agent of bacterial twig dieback of Corylus avellana

Thirty-eight bacterial strains isolated from hazelnut (Corylus avellana) cv. Tonda Gentile delle Langhe showing a twig dieback in Piedmont and Sardinia, Italy, were studied by a polyphasic approach. All strains were assessed by fatty acids analysis and repetitive sequence-based polymerase chain reaction (PCR) fingerprinting using BOX and ERIC primer sets. Representative strains also were assessed by sequencing the 16S rDNA and hrpL genes, determining the presence of the syrB gene, testing their biochemical and nutritional characteristics, and determining their pathogenicity to hazelnut and other plants species or plant organs. Moreover, they were compared with reference strains of other phytopathogenic pseudomonads. The strains from hazelnut belong to Pseudomonas syringae (sensu latu), LOPAT group Ia. Both fatty acids and repetitive-sequence-based PCR clearly discriminate such strains from other Pseudomonas spp., including P. avellanae and other P. syringae pathovars as well as P. syringae pv. syringae strains from hazelnut. Also, the sequencing of 16S rDNA and hrpL genes differentiated them from P. avellanae and from P. syringae pv. syringae. They did not possess the syrB gene. Some nutritional tests also differentiated them from related P. syringae pathovars. Upon artificial inoculation, these strains incited severe twig diebacks only on hazelnut. Our results justify the creation of a new pathovar because the strains from hazelnut constitute a homogeneous group and a discrete phenon. The name of P. syringae pv. coryli is proposed and criteria for routine identification are presented.

Pseudomonas syringae pv. phaseolicola: from ‘has bean’ to supermodel

Pseudomonas syringae pv. phaseolicola causes halo blight of the common bean, Phaseolus vulgaris, worldwide and remains difficult to control. Races of the pathogen cause either disease symptoms or a resistant hypersensitive response on a series of differentially reacting bean cultivars. The molecular genetics of the interaction between P. syringae pv. phaseolicola and bean, and the evolution of bacterial virulence, have been investigated in depth and this research has led to important discoveries in the field of plant-microbe interactions. In this review, we discuss several of the areas of study that chart the rise of P. syringae pv. phaseolicola from a common pathogen of bean plants to a molecular plant-pathogen supermodel bacterium. Taxonomy: Bacteria; Proteobacteria, gamma subdivision; order Pseudomonadales; family Pseudomonadaceae; genus Pseudomonas; species Pseudomonas syringae; Genomospecies 2; pathogenic variety phaseolicola. Microbiological properties: Gram-negative, aerobic, motile, rod-shaped, 1.5 µm long, 0.7-1.2 µm in diameter, at least one polar flagellum, optimal temperatures for growth of 25-30 °C, oxidase negative, arginine dihydrolase negative, levan positive and elicits the hypersensitive response on tobacco. Host range: Major bacterial disease of common bean (Phaseolus vulgaris) in temperate regions and above medium altitudes in the tropics. Natural infections have been recorded on several other legume species, including all members of the tribe Phaseoleae with the exception of Desmodium spp. and Pisum sativum. Disease symptoms: Water-soaked lesions on leaves, pods, stems or petioles, that quickly develop greenish-yellow haloes on leaves at temperatures of less than 23 °C. Infected seeds may be symptomless, or have wrinkled or buttery-yellow patches on the seed coat. Seedling infection is recognized by general chlorosis, stunting and distortion of growth. Epidemiology: Seed borne and disseminated from exudation by water-splash and wind occurring during rainfall. Bacteria invade through wounds and natural openings (notably stomata). Weedy and cultivated alternative hosts may also harbour the bacterium. Disease control: Some measure of control is achieved with copper formulations and streptomycin. Pathogen-free seed and resistant cultivars are recommended. Useful websites: Pseudomonas-plant interaction http://www.pseudomonas-syringae.org/; PseudoDB http://xbase.bham.ac.uk/pseudodb/; Plant Associated and Environmental Microbes Database (PAMDB) http://genome.ppws.vt.edu/cgi-bin/MLST/home.pl; PseudoMLSA Database http://www.uib.es/microbiologiaBD/Welcome.html.

Miniature transposable sequences are frequently mobilized in the bacterial plant pathogen Pseudomonas syringae pv. phaseolicola

Mobile genetic elements are widespread in Pseudomonas syringae, and often associate with virulence genes. Genome reannotation of the model bean pathogen P. syringae pv. phaseolicola 1448A identified seventeen types of insertion sequences and two miniature inverted-repeat transposable elements (MITEs) with a biased distribution, representing 2.8% of the chromosome, 25.8% of the 132-kb virulence plasmid and 2.7% of the 52-kb plasmid. Employing an entrapment vector containing sacB, we estimated that transposition frequency oscillated between 2.661025 and 1.161026, depending on the clone, although it was stable for each clone after consecutive transfers in culture media. Transposition frequency was similar for bacteria grown in rich or minimal media, and from cells recovered from compatible and incompatible plant hosts, indicating that growth conditions do not influence transposition in strain 1448A. Most of the entrapped insertions contained a full-length IS801 element, with the remaining insertions corresponding to sequences smaller than any transposable element identified in strain 1448A, and collectively identified as miniature sequences. From these, fragments of 229, 360 and 679-nt of the right end of IS801 ended in a consensus tetranucleotide and likely resulted from one-ended transposition of IS801. An average 0.7% of the insertions analyzed consisted of IS801 carrying a fragment of variable size from gene PSPPH_0008/PSPPH_0017, showing that IS801 can mobilize DNA in vivo. Retrospective analysis of complete plasmids and genomes of P. syringae suggests, however, that most fragments of IS801 are likely the result of reorganizations rather than one-ended transpositions, and that this element might preferentially contribute to genome flexibility by generating homologous regions of recombination. A further miniature sequence previously found to affect host range specificity and virulence, designated MITEPsy1 (100-nt), represented an average 2.4% of the total number of insertions entrapped in sacB, demonstrating for the first time the mobilization of a MITE in bacteria.

Hcp2, a secreted protein of the phytopathogen Pseudomonas syringae pv. tomato DC3000, is required for competitive fitness against bacteria and yeasts

When analysing the secretome of the plant pathogen Pseudomonas syringae pv. tomato (Pst) DC3000, we identified hemolysin co-regulated protein (Hcp) as one of the secreted proteins. Hcp is assumed to be an extracellular component of the type VI secretion system (T6SS). Two copies of hcp genes are present in the Pst DC3000 genome, hcp1 (PSPTO_2539) and hcp2 (PSPTO_5435). We studied the expression patterns of hcp genes and tested the fitness of hcp knock-out mutants in host plant colonization and in inter-microbial competition. We found that the hcp2 gene is expressed, most actively at the stationary growth phase, and that the Hcp2 protein is secreted via T6SS and appears in the culture medium as covalently linked dimers. Expression of hcp2 is not induced in planta and it does not contribute to virulence or colonisation in tomato or Arabidopsis plants. Instead, hcp2 is required for survival in competition with enterobacteria and yeasts, and its function is associated with suppression of the growth of these competitors. This is the first report on bacterial T6SS-associated genes functioning in competition against yeast. Our results suggest that the T6SS of P. syringae may play an important role in bacterial fitness, allowing this plant pathogen to survive in conditions where it has to compete with other micro-organisms for resources.

Pseudomonas aeruginosa elastase disables proteinase-activated receptor 2 in respiratory epithelial cells

Pseudomonas aeruginosa, a major lung pathogen in cystic fibrosis (CF) patients, secretes an elastolytic metalloproteinase (EPa) contributing to bacterial pathogenicity. Proteinase-activated receptor 2 (PAR2), implicated in the pulmonary innate defense, is activated by the cleavage of its extracellular N-terminal domain, unmasking a new N-terminal sequence starting with SLIGKV, which binds intramolecularly and activates PAR2. We show that EPa cleaves the N-terminal domain of PAR2 from the cell surface without triggering receptor endocytosis as trypsin does. As evaluated by measurements of cytosolic calcium as well as prostaglandin E(2) and interleukin-8 production, this cleavage does not activate PAR2, but rather disarms the receptor for subsequent activation by trypsin, but not by the synthetic receptor-activating peptide, SLIGKV-NH(2). Proteolysis by EPa of synthetic peptides representing the N-terminal cleavage/activation sequences of either human or rat PAR2 indicates that cleavages resulting from EPa activity would not produce receptor-activating tethered ligands, but would disarm PAR2 in regard to any further activating proteolysis by activating proteinases. Our data indicate that a pathogen-derived proteinase like EPa can potentially silence the function of PAR2 in the respiratory tract, thereby altering the host innate defense mechanisms and respiratory functions, and thus contributing to pathogenesis in the setting of a disease like CF.

Association of lipoprotein lipase gene polymorphisms with obesity and type 2 diabetes in an Asian Indian population

AIMS: Lipoprotein lipase (LPL), a pivotal enzyme in lipoprotein metabolism, catalyzes the hydrolysis of triglycerides of very low-density lipoproteins and chylomicrons. Assuming that the variants in the promoter of the LPL gene may be associated with changes in lipid metabolism leading to obesity and type 2 diabetes, we examined the role of promoter variants (-T93G and -G53C) in the LPL gene in an urban South Indian population. METHODS: The study subjects (619 type 2 diabetic and 731 normal glucose-tolerant (NGT) subjects) were chosen from the Chennai Urban Rural Epidemiology Study, an ongoing population-based study in southern India. The polymorphisms were genotyped using polymerase chain reaction-restriction-fragment length polymorphism (PCR-RFLP). Linkage disequilibrium (LD) was estimated from the estimates of haplotypic frequencies. RESULTS: The two polymorphisms studied were not in LD. The -T93G was not associated with type 2 diabetes but was associated with obesity. 11.5% of the obese subjects (62/541) had the XG(TG+GG) genotype compared with 6.4% of the nonobese subjects (52/809; P=0.001). The odds ratio for obesity for the XG genotype was 1.766 (95% CI: 1.19-2.63, P=0.005). Subjects with XG genotype also had higher body mass index and waist circumference compared with those with TT genotype. With respect to G53C, subjects with the XC(GC+CC) genotype had 0.527 and 0.531 times lower risk for developing type 2 diabetes and obesity, respectively. CONCLUSIONS: Among Asian Indians, the -T93G SNP of the LPL gene is associated with obesity but not type 2 diabetes, whereas the -G53C SNP appears to be protective against both obesity and type 2 diabetes.

Draft genome sequence of pseudomonas corrugata, a phytopathogenic bacterium with potential industrial applications

Pseudomonas corrugata was first described as the causal agent of a tomato disease called 'pith necrosis' yet it is considered as a biological resource in various fields such as biocontrol of plant diseases and production of industrially promising microbial biopolymers (mcl-PHA). Here we report the first draft genome sequence of this species.

Bdellovibrio bacteriovorus HD100 guards against Pseudomonas tolaasii brown-blotch lesions on the surface of post-harvest Agaricus bisporus supermarket mushrooms

BACKGROUND: Pseudomonas tolaasii is a problematic pathogen of cultured mushrooms, forming dark brown 'blotches' on mushroom surfaces and causing spoilage during crop growth and post-harvest . Treating P. tolaasii infection is difficult, as other, commensal bacterial species such as Pseudomonas putida are necessary for mushroom growth, so treatments must be relatively specific. RESULTS: We have found that P. tolaasii is susceptible to predation in vitro by the δ-proteobacterium Bdellovibrio bacteriovorus. This effect also occurred in funga, where B. bacteriovorus was administered to post-harvest mushroom caps before and after administration of the P. tolaasii pathogen. A significant, visible improvement in blotch appearance, after incubation, was observed on administration of Bdellovibrio. A significant reduction in viable P. tolaasii cell numbers, recovered from the mushroom tissue, was detected. This was accompanied by a more marked reduction in blotch severity on Bdellovibrio administration. We found that there was in some cases an accompanying overgrowth of presumed-commensal, non-Pseudomonas bacteria on post-harvest mushroom caps after Bdellovibrio-treatment. These bacteria were identified (by 16SrRNA gene sequencing) as Enterobacter species, which were seemingly resistant to predation. We visualised predatory interactions occuring between B. bacteriovorus and P. tolaasii on the post-harvest mushroom cap surface by Scanning Electron Microscopy, seeing predatory invasion of P. tolaasii by B. bacteriovorus in funga. This anti-P. tolaasii effect worked well in post-harvest supermarket mushrooms, thus Bdellovibrio was not affected by any pre-treatment of mushrooms for commercial/consumer purposes. CONCLUSIONS: The soil-dwelling B. bacteriovorus HD100 preys upon and kills P. tolaasii, on mushroom surfaces, and could therefore be applied to prevent spoilage in post-harvest situations where mushrooms are stored and packaged for sale.

The permeability parameter of the outer membrane of pseudomonas aeruginosa Varies with the concentration of a test substrate, cephalosporin C

The permeability parameter (C) for the movement of cephalosporin C across the outer membrane of Pseudomonas aeruginosa was measured using the widely accepted method of Zimmermann & Rosselet. In one experiment, the value of C varied continuously from 4·2 to 10·8 cm3 min-1 (mg dry wt cells)-1 over a range of concentrations of the test substrate, cephalosporin C, from 50 to 5 μm. Dependence of C on the concentration of test substrate was still observed when the effect of a possible electric potential difference across the outer membrane was corrected for. In quantitative studies of β-lactam permeation the dependence of C on the concentration of β-lactam should be taken into account.

Selection experiments reveal trade-offs between swimming and twitching motilities in pseudomonas aeruginosa

Bacteria possess a range of mechanisms to move in different environments, and these mechanisms have important direct and correlated impacts on the virulence of opportunistic pathogens. Bacteria use two surface organelles to facilitate motility: a single polar flagellum, and type IV pili, enabling swimming in aqueous habitats and twitching along hard surfaces, respectively. Here, we address whether there are trade-offs between these motility mechanisms, and hence whether different environments could select for altered motility. We experimentally evolved initially isogenic Pseudomonas aeruginosa under conditions that favored the different types of motility, and found evidence for a trade-off mediated by antagonistic pleiotropy between swimming and twitching. Moreover, changes in motility resulted in correlated changes in other behaviors, including biofilm formation and growth within an insect host. This suggests environmental origins of a particular motile opportunistic pathogen could predictably influence motility and virulence

Competition and dispersal in Pseudomonas aeruginosa

Dispersal plays a crucial role in a range of evolutionary and ecological processes; hence there is strong motivation to understand its evolution. One key prediction is that the relative benefits of dispersal should be greater when dispersing away from close relatives, because in this case dispersal has the additional benefit of alleviating competition with individuals who share the same dispersal alleles. We tested this prediction for the first time using experimental populations of the opportunistic pathogen Pseudomonas aeruginosa. We measured the fitness of isogenic genotypes that differed only in their dispersal behaviors in both clonal and mixed populations. Consistent with theory, the benefit of dispersal was much higher in clonal populations, and this benefit decreased with increasing growth rate costs associated with dispersal.

Functionality of lipase and emulsifiers in low-fat cakes with inulin

The functional effects of lipase (0.003 and 0.006 g/100 g of flour) and emulsifier (0.5 and 1 g/100 g of flour) on fat-replaced (0%, 50% and 70%) batters and cakes with inulin (0, 7.5 and 10 g/100 g/of flour, respectively) were studied. Emulsifier addition significantly lowered the relative density of the batter. Emulsifier incorporation increased the viscoelastic properties of the batter. In contrast, lipase incorporation decreased the degree of system structuring. The evolution of the dynamic moduli and complex viscosity with rising temperatures were studied. Batters with 1 g/100 g emulsifier displayed a significantly lower complex viscosity during heating, resulting in collapsed cakes. Differential scanning calorimetry results revealed that the thermal setting in the control cakes occurred at higher temperatures, and accordingly, greater cake expansion was observed. Cakes with 0.003 g/100 g lipase or 0.5 g/100 g emulsifier displayed volume and crumb cell structure that were similar to those of control cakes. Higher concentrations of both improvers gave rise to cakes with lower volume, higher hardness and lower springiness. During storage time, cakes with lipase displayed lower hardness. Both improvers, at low concentrations, could improve certain physical characteristics, such as crumb structure, of fat-replaced cakes with inulin.